Prophylaxis and therapy of acquired immunodeficiency syndrome

ABSTRACT

An active peptide consisting essentially of 7 to about 30 residue and having a sequence that corresponds to a conserved domain of an HIV protein is disclosed, as is a multimer containing that peptide, an aqueous composition containing the multimer and methods of using and making the same. The aqueous composition containing an immunologically effective amount of an active peptide multimer, when introduced into an immunocompetent host animal in an immunologically effective amount, is capable of inducing cellular immunity against the native HIV protein to which the active peptide of the multimer corresponds in sequence, but is not capable of inducing production of antibodies that immunoreact with that native HIV protein.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a division of application Ser. No. 07/410,727 filedSep. 20, 1989 now U.S. Pat. No. 5,128,319, which was acontinuation-in-part of Ser. No. 07/090,646, filed Aug. 28, 1987, nowabandoned.

FIELD OF THE INVENTION

The present invention concerns a method to prevent or treat acquiredimmunodeficiency syndrome (AIDS) and involves a new and novel approachfor making an immunizing composition or inoculum. The inoculum orcomposition comprises synthetic peptide multimer that exhibits certain Tcell activating immunological characteristics of one or more proteinsencoded by the viral causative agent of this disease.

BACKGROUND OF THE INVENTION

AIDS was first recognized in the United States in 1981; the number ofcases has been increasing at a dramatic pace since then. Since 1978 morethan 2.4 million AIDS infections have been reported in the UnitedStates, alone (Rees, Nature, 326:343, 1987). Once significantimmunosuppressive symptoms appear in an infected individual, theexpected outcome of the infection is death. There is currently no knowntreatment that can indefinitely delay or prevent the fatal consequencesof the disease. Although the disease first manifested itself inhomosexual or bisexual males and intravenous drug abusers, it has nowspread to others by means such as intimate sexual contact with orreceipt of blood products from a carrier of the virus.

The causative agent, associated with AIDS has been identified as a groupof closely related retroviruses commonly known as Human T CellLymphotrophic Virus-type III (HTLV-III), Lymphadenopathy Viruses (LAV),AIDS-Related Viruses (ARV), or more recently named HumanImmunodeficiency Virus (HIV). These viruses will be collectivelyreferred to herein for convenience as HIV.

Like other retroviruses, HIV has RNA as its genetic material. When thevirus enters the host cell, a viral enzyme known as reversetranscriptase copies the viral RNA into a double stranded DNA. The viralDNA migrates to the nucleus of the cell where it serves as a templatefor additional copies of viral RNA which can then be assembled into newviral particles. The viral RNA can also serve as messenger RNA forcertain viral proteins [either the viral core proteins (known as p18,p24 and p13)] or the reverse transcriptase, or be “spliced” intospecific viral messenger RNAs necessary to produce several other viralproteins including two glycosylated structural proteins known as gp41and gp120 which are inserted in the outer membrane of the virus(Wain-Hobson et al., Cell 40:9, 1985). A recent study has shown thatpurified gp120 induces antibody in the goat, horse and rhesus monkeythat neutralizes HIV in lab tests (Robey et al., Proc. Natl. Acad. Sci.,USA 83:7023, 1986).

Vaccines have been used for many years to prevent infections caused byagents such as viruses. The general approach has been to inject healthyindividuals with, for example, a killed or modified virus preparation inorder to prime the individual's immune systems to mount an assault onthe infecting virus. Recent advances in recombinant DNA technology haveallowed safer methods of vaccination that involve use of exposed viralcomponents produced by microbial systems. After sufficient purification,the viral component, for example a protein subunit, is administered as avaccine in a suitable vehicle and/or an adjuvant. The latter stimulatesthe host's system in a way that improves the immune response to theviral subunit.

Another potential method of making a vaccine is by using chemicallysynthesized peptide fragments of a viral protein subunit. This methodhas several advantages over the other methods of producing vaccines,including purity of the product, reproducibility and specificity of theimmune response.

Surface antigens of an infecting virus can elicit T cell and B cellresponses. From the work of Milich and coworkers (Milich et al., J. Exp.Med. 164:532, 1986; Milich and McLachlan, Science, 232:1398, 1986) it isclear that some regions of a protein's peptide chain can possess eitherT cell or B cell epitopes. These epitopes are frequently distinct fromeach other and can comprise different peptide sequences. Other examplesinclude the work of Maizel et al., (Eur. J. Immunol. 10:509, 1980) forhen egg-white lysozyme, and Senyk et al., (J. Exp. Med., 133:1294, 1971)for glucagon. Thus, short stretches of a protein sequence can elicit a Tcell response but not a B cell response. A more complete review of theseand other observations pertinent to this point is included in the workof Livingstone and Fathman (Ann. Rev. Immunol., 5:477, 1987).

A short peptide region within the surface protein of infectiousHepatitis B virus has been shown to elicit only a T cell response inmice (Milich et al., 1986). Specifically, a synthetic peptide, whosesequence is derived from amino acids numbered 120-132 located within thepre-S(2) domain of the Hepatitis B surface antigen gene, elicited a verystrong T cell priming response to the peptide but stimulated only a veryweak antibody response. In other words, mice mounted a poor antibodyresponse to that peptide, but the T cells of immunized mice wereefficiently primed (i.e. activated) to recognize that peptide asmeasured in T cell proliferation assays (Milich et al., 1986). The lowlevel of the antibody produced by mice immunized with this peptide didnot bind to the native viral surface antigen. The sequence of this Tcell active peptide is:

Amino terminal-MQWNSTTFHQTLQ-carboxy-terminal. The single letter codefor amino acids used throughout this application is: A, alanine; Ccysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G,glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M,methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S,serine; T, threonine; V, valine; W, tryptophan; and Y, tyrosine.

In contrast to the above-described results, a second peptide sequence(amino acids 132-145) elicited a very weak T-cell response in mice(Milich et al., 1986). This second peptide did, however, efficientlybind antibody raised against it under conditions where a T cell epitopeis provided.

The sequence of the second or B cell active peptide is:

Amino terminal-DPRVRGLYFPAGG-carboxy-terminal. Mice were also immunizedwith a longer peptide made up of both of the above-mentioned T- andB-active peptide sequences. In this case, high titers of antibody wereproduced against the B site peptide but not the T site peptide. Thecombination of both T- and B-sites within one peptide should stimulateboth T and B cell responses, as measured by producing a specificantibody to the B cell epitope of the peptide chain. Synthetic peptideantigens may be constructed to produce two types of immune responses:T-cell only and T cell combined with a B cell response.

Cellular immune responses provide a major mechanism for reducing thegrowth of virus-infected cells (Doherty et al., Adv. Cancer Res., 42:1,1985). A report by Earl et al., (Science, 234:728, 1986) demonstratedT-lymphocyte priming and protection against the Friend virus (aretrovirus)-induced mouse leukemia by a viral surface protein vaccine.Direct evidence for the role of a subset of T-lymphocytes (OKT8/LEU2positive) in suppressing HIV growth in vitro was recently obtained byWalker et al., (Science, 234:1563, 1986). This study furtherdemonstrated that, after depletion of CD8⁺ T-lymphocytes from the bloodof HIV-infected individuals, large quantities of HIV were isolated fromperipheral blood mononuclear cells of four of seven asymptomatic,seropositive homosexual men who were initially virus-negative or hadvery low levels of virus. Thus, the CD8⁺ subset of T-lymphocytes mayplay a role in virus infected individuals to prevent HIV replication anddisease progression.

SUMMARY OF THE INVENTION

The present invention contemplates a peptide, a peptide multimer, anaqueous composition containing the peptide multimer and a method ofusing the composition.

A peptide of the invention contains 7 to about 30 amino acid residues,and has a sequence that corresponds to a conserved domain of an HIVprotein such as the gp160 envelope and core proteins. Preferred peptideshave a sequence that corresponds to a portion of a conserved domainselected from the group consisting of the first, second, third and fifthconserved domains of the gp160 molecule.

A peptide of the invention is generally used as a portion of a peptidemultimer. Two specific classes of peptide multimers are disclosed. Inone class, the amino-terminal residue of a peptide is peptide-bonded toa spacer peptide that contains an amino-terminal lysyl residue and oneto about five amino acid residues such as glycyl residues to form acomposite polypeptide. Those added residues of the spacer peptide do notinterfere with the immunizing capacity of the multimer, nor with itscapacity to form surfactant-like micelles in aqueous compositions. Thealpha- and epsilon-amino groups of the amino-terminal lysyl residue areamidified with a C₁₂-C₁₈ fatty acid such as palmitic acid to form thereaction product that is used. The di-amide so formed formssurfactant-like micellular multimers in an aqueous composition.

A second class of multimer is a polymer having a before-describedpeptide as a repeating unit. Here, each peptide is synthesized tocontain a cysteine (Cys) residue at each of its amino- andcarboxy-termini. The resulting di-cysteine-terminated (di-Cys) peptideis then oxidized to polymerize the di-Cys peptide monomers into apolymer or cyclic peptide multimer in which the peptide repeating unitsare linked by stine (oxidized cysteine) residues.

A peptide multimer of either class can contain one or a plurality ofdifferent peptide sequences. A before-described peptide of a multimer isan “active” peptide in that when used in a composition discussed below,the multimer can induce cell mediated immunity such as production ofcytotoxic T cells. A multimer can also include an inactive peptide, forexample to assist in dispersing the multimer in the aqueous medium. Thelysyl-containing peptide spacer discussed before can be viewed as suchan inactive peptide.

The peptide multimer is utilized in an aqueous composition (inoculum).That composition contains water having a before-described multimerdispersed therein. The composition, when used to immunize animmunocompetent host animal such as a mouse, has the capacity ofinducing cell mediated immunity such as cytotoxic T cell activation tothe native HIV protein corresponding in sequence to that of an activepeptide of the multimer, but does not substantially induce production ofantibodies that immunoreact with that corresponding native HIV protein.The composition thus contains an immunizing effective amount of abefore-discussed multimeric peptide.

In one method aspect of the invention, an immunizing amount of an abovecomposition containing an immunizing effective amount of an activepeptide multimer is introduced into (administered to) an animal hostsuch as a mouse or human to induce cellular immunity such as T cellimmunity to a preselected native HIV protein without production ofantibodies that immunoreact with that preselected native HIV protein.The preselected HIV protein is the HIV protein to which the activepeptide corresponds in sequence. The immunized animal is then maintainedto permit the immunity to be induced. This immunization can be repeatedor boosted as desired.

Another method aspect of this invention is a method of killing targetcells that exhibit an HIV protein or a portion of an HIV protein on thecell surfaces. Here, target cells that exhibit an HIV protein or aportion of an HIV protein on their cell surfaces such as HIV-infected Tcells or leukocytes that are artificially made to express cell surfaceHIV proteins are contacted with a killing effective amount of cytotoxicT cells that have been activated using a before-described composition.The cell surface-exhibited HIV protein and the HIV protein to which anactive peptide of the multimer corresponds in sequence are the sameproteins, since the core protein and the two processed portions of thegp160 protein (the gp120 and gp41 envelope proteins) are the proteinsnormally found on HIV-infected cell surfaces. That contact is maintainedfor a time period sufficient for the cytotoxic T cells to kill thetarget cells. This method can be carried out in vitro or in viva in thebody of a host animal.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings forming a portion of this disclosure,

FIG. 1 is a graph that illustrates the in vitro proliferation ofpopliteal lymph node (PLN) cells after in vivo immunization of Balb/cmice with an aqueous composition containing an immunologically effectiveamount of a peptide multimer polymer of this invention prepared fromeach of peptides 61, 63, 65 and 67. Tuberculin purified proteinderivative (PPD) was used as a control, as shown. A ³H-thymidine(³H-TdR) incorporation assay was used for these studies. The data areillustrated as a stimulation index, which is calculated as the foldincrease in radioactivity counts in the presence of the peptide multimerantigen compared to background values where no antigen was added.Details of this study are discussed hereinafter.

FIG. 2 is a graph that illustrates ³H-TDR incorporation (T cellproliferation) of PLN cells after immunization of B6C3 F1 mice with anaqueous composition containing an immunologically effective amount of apeptide multimer polymer of this invention prepared from each ofpeptides 61, 63, 65 and 67. An unrelated peptide, PPD and gp160 wereused as controls. The data are shown as the ³H-TdR incorporation [delta(Δ) counts per minute (cpm)] obtained by subtracting radioactivityvalues in control wells without added antigen from those in wells withantigen. Details of this study and those of the studies of FIGS. 3-5 arediscussed hereinafter.

FIG. 3 is a graph similar to that of FIG. 2 except A.SWxBalb/c F1 micewere utilized as the animal hosts.

FIG. 4 is a graph similar to that of FIG. 2 in which multimers preparedfrom peptides 103 through 117 (a through o, respectively) were used toimmunize B6C3 F1 mice.

FIG. 5 is a graph similar to that of FIG. 4 except that A.SWxBalb/c micewere again used as the animal hosts.

FIGS. 6A-6B contains two panels of graphs that illustrate B6C3 F1 mousePLN cell proliferation by ³H-TdR incorporation as described before usingvarying concentrations of peptide multimer and gp120 as antigens. PanelA illustrates results for a peptide multimer polymer prepared frompeptides 104, whereas Panel B illustrates results using a multimerprepared from peptide 106. PPD and unrelated peptide were used ascontrols. A further discussion relating to FIGS. 6-8 is foundhereinafter.

FIGS. 7A-7B contain two panels, and illustrates studies of PLN cellproliferation from B6C3 F1 mice using various concentrations of gp160and peptide multimer polymers prepared from peptides 61 (Panel A) and 63(Panel B) as antigens, with PPD, and an unrelated peptide as controls.

FIGS. 8A-8B contain two panels, and illustrates studies of PLN cellproliferation from B6C3 F1 mice using various concentrations of gp120and peptide multimer polymers prepared from peptides 65 (Panel A) and111 (Panel B) as antigens, with PPD and an unrelated peptide ascontrols.

DESCRIPTION OF THE PREFERRED EMBODIMENT

A. Overview

The HIV agent is unique in that it infects cells involved in the immuneresponse and can kill these cells. The host cell often involved is theT4 lymphocyte, a white blood cell that plays a central role inregulating the immune system. The virus binds to cell surface T4 proteinwhich is implicated in the mediation of efficient T cell-target cellinteractions. T4⁺ lymphocytes interact with target cells expressingmajor histocompatibility (MHC) class II gene products.

Both T4 and MHC genes are members of the immunoglobulin gene family(Maddon et al., Cell, 47:333, 1986). The observation that T4 interactswith the exterior HIV envelope protein, gp120, prompted a structuralcomparison of the viral protein to immunoglobulin proteins.Interestingly, two regions of gp120 were found to share sequencehomology with human immunoglobulin heavy chain constant regions (Maddonet al., Cell, 47:333, 1986).

Extrapolating from these observations, the present invention hinges tosome extent upon the fact that gp120 has certain properties unique tohuman immunoglobulins. Furthermore, this similarity in structure mayallow the virus to escape inactivation by antibody interaction. Stillfurther, viral-antibody interaction may, in certain situations, increasethe infectivity of the virus.

For example, recent work suggests that AIDS patients can and do haveantibodies that neutralize the virus, as determined by in vitrolaboratory tests. Yet these same patients die of the disease.

The present invention contemplates that antibodies binding to the virusmay not interfere with and in some cases may even increase the virus'inherent ability to infect the patient's lymphoid cells. Recently,retrovirus infectivity was shown to be increased by binding ofanti-retrovirus antibodies (Legrain et al., J. Virol., 60:1141, 1986).Therefore, an AIDS vaccine that primes the individual's immune system tomake antibodies to viral surface proteins may enhance the infectivity ofan already deadly virus. What is needed then is to stimulate only theindividual's T cell immunity (for example, cytotoxic T cells or CD8⁺ Tcells) without involving an antibody response to viral proteins.Synthetic peptide immunogens can certainly achieve this result.

B. The Peptides

A peptide useful herein consists essentially of no more than about 30,and more preferably about 10 to about 25, amino acid residues and has asequence that corresponds to a portion of a conserved domain of an HIVprotein such as the gp120 envelope, gp41 envelope and core proteins. Thegp41 and gp120 envelope proteins are portions of the precursor gp160envelope protein.

It is therefore often convenient to refer to portions of the gp160protein rather than the gp41 and gp120 molecules. Indeed, particularlypreferred peptides have sequences that correspond to sequences of thefirst, second, third and fifth conserved domains of gp160, with thefifth domain being in the gp4l portion of the processed gp160 protein.

A useful peptide most preferably contains only those amino acid residuesthat are identical or homologous to (conservative substitutions for)residues present in a corresponding sequence of a conserved domain of anHIV protein. An additional number of residues of substantially anylength can also be present at either or both termini of the peptide, upto a total of about 30 residence in a peptide. However, any additionalresidues must not interfere with the activity of the peptide, such asits ability to activate cytotoxic T cells and its substantial inabilityto induce production of antibodies that immunoreact with thecorresponding native protein. Therefore, a peptide of this invention issaid to “consist essentially” of an enumerated sequence.

The phrase “corresponds” as used herein means that an amino acid residuesequence has the same linear arrangement of the same amino acid residuesas the sequence of the conserved HIV protein domain to which it“corresponds”. However, it is well-known in the art that substitutionsfor amino acid residues can be made that are equivalent immunogenically.

In particular, conservative substitutions such as one hydrophobic orpolar residue for another at one or more of many positions in a peptidefrequently does not alter the immunogenic characteristics of thepeptide. For example, an aspartic acid residue can be exchanged for aglutamic acid residue, or a leucine residue exchanged for an isoleucine.Thus, exchanges that destroy the amphipathic character of a peptide areexcluded.

A first step in preparation of a peptide of the present invention is toprepare a number of peptides containing 7 to about 30, and preferablyabout 10 to about 25, amino acid residues in length and having an aminoacid sequence that corresponds to a conserved domain of an HIV protein.For example, a large portion of gp41 is conserved among the sevenstrains of HIV-sequenced to date (Modrow et al., J. Virol., 61:570,1987).

Computer programs have been developed that are useful in predicting Tcell recognition sites and antibody binding sites within antigens (thelatter known as B cell sites). Several computer programs can be usedsuch as the be Lisi and Berzofsky program for T cell sites (Proc. Natl.Acad. Sci. USA, 82:7048, 1985), and for B cells - the Hopp and Woodsprogram (J. Mol. Biol., 157:105, 1982) and the Sette et al., program(Mol. Immunol., 23:807, 1986. Short synthetic peptides were made frompredicted T cell regions.

Using the computer program of Sette et al., (1986) to analyze the linearsequence of the HIV envelope proteins, several proposed T cell epitopeswere selected from a first conserved segment of gp120 (Modrow et al., J.Virol., 61:570-578) as illustrative examples. Their sequences are asfollows the amino-terminus at the left and carboxy-terminus on theright, in standard manner:

(1) CSAVEQLWVTVY;

(2) TTLFCASDAKAY;

(3) EVVLGNVTENFNM;

(4) QMHEDIISLWDQS; and

(5) QSLKPCVKLTPLC.

These peptides are predicted T cell epitopes within a 100 amino acidstretch of conserved sequences near the amino terminus of the gp120protein. A recent report indicated that this region is active instimulating T cell immunity (Ahearne et al., III InternationalConference on AIDS, held in Washington, D.C., Jun. 1-5, 1987, abstract #M.10.3, page 8).

Antigenic sites recognized by T cells have been reported to correlatewith helical structures (either alpha helices or another type helixcalled a 3₁₀ helical structure). Such antigenic sites are also thoughtto be protein segments displaying a polar/apolar character, forming astable amphipathic structure with separated hydrophobic and hydrophilicsurfaces and/or protein segments displaying a marked change inhydrophilicity between the first-half and the second-half of a block ofamino acids (differential amphipathic structures).

In practice, using computer programs, the helical structures areidentified by a consistent stretch of blocks of amino acids (each blockbeing 6-7 residues in length) with angles (termed delta values) of100°±20° (alpha helix) or 120°±15° (3₁₀ helical structure). Differentialamphipathic structures are identified by peaks of differentialhydrophilicity (See Table 1). For the purpose of selecting regions thatare predicted to be poor antibody eliciting and/or binding sites, thesestructures should have negative mean hydrophilicity values. All of thesevalues are listed below in Table 1 as the computer analysis of aconserved gp120 sequence (residues 35-137).

TABLE 1 ANALYSIS OF AN HIV-CONSERVED AMINO ACID SEQUENCE REGIONSEQUENCE:CSAVEQLWVTVYYCVPVWKEATTTLFCASDAKAYSTEVHNVWATHACVPTDPNPQEVVLCNVTENFNMWKNNMVEQMHEDIIISLWDQSLKPCVKLTPLC BLOCK LENGTH 6 BLOCK LENGTH 7 HOPP/WOODSSCALE KYTE/DOOLITTLE SCALE MEAN HYDROPHILICITY = .0356494236 MEANHYDROPHILICITY = .0292929294 MEAN DIFF. HYDR. = 4.72234043 MEAN DIFF.HYDR. = 5.527957 STANDARD DEV. = .41640836 STANDARD DEV. = .436292101amphipsthicity amphipathicity Block hydrophilicity peaks Blockhydrophilicity peaks No. seq. mean diff. angle amph valve No. seq. meandiff. angle amph valve  1 CSAVEQ .08 2.9 130 5.2603542  1 CSAVEQL −.656.7 110 13.7622165 180 4.05713223  2 SAVEQL −.05 3.1 106 6.07139973  2SAVEQLW −.16 5.8 106 12.8605401  3 AVEQLW .46 .8 118 8.61223123  3AVEQLWV −.88 4.6 80 8.87890778 135 11.9637599  4 VEQLWV .3 1.6 1349.74138842  4 VEQLWVT −.52 5.4 80 9.54449739 143 13.3879542  5 EQLWVT.49 .1 123 8.21074778  5 EQLWVTV −.52 10.9 91 8.24956064 180 13.2857142 6 QLWVTV −.27 5.2 84 3.68722078  6 QLWVTVY −.83 2.8 108 6.12546694 1807.99999744 180 19.4265565  7 LWVTVY −.69 4.3 80 3.40803117  7 LWVTVYY−1.15 5.5 88 5.78042364 145 6.79282004 180 12.6571412  8 WVTVYY −.77 7.6117 5.2587283  8 WVTVYYG −.55 5.6 80 3.90743985 180 5.99999683 18010.9428538  9 VTVYYG −1.61 2.8 80 2.27261392  9 VTVYYGV −1.28 5.2 805.68423569 180 .999999507 112 7.47691013 180 12.428560L 10 TVYYGV −1.611.2 80 2.36217496 10 TVYYGVP −.45 0 100 10.086216L 180 .999999844 18011.5428464 11 VYYGVP −1.54 3 80 1.85508868 11 VYYGVPV −1.15 5.2 1119.23573655 144 1.91242888 180 13.4571299 12 YYGVPV −1.54 3.2 801.8054094 12 YYGVPVW −.42 4.7 80 2.88605369 144 1.85463866 18011.7142808 13 YGVPVW −.59 7.3 113 4.82991766 13 YGVPVWK −.05 3.1 804.28917765 180 7.09999547 132 11.3842845 14 GVPVWK .29 8 80 3.4621416314 GVPVWKE .27 10.5 80 5.05560131 122 5.65042609 118 7.81868378 18010.6285662 15 VPVWKE 1.06 12.4 80 4.21156022 15 VPVWKEA −.05 12.4 806.62653296 180 6.39999629 112 9.64156172 180 12.2571317 16 PVWKEA 1.233.6 80 7.35037792 16 PVWKEAT .65 4.1 87 11.1679208 180 5.39999818 1808.14285043 17 VWKEAT 1.16 2.8 80 7.51038801 17 VWKEATT .52 1 8311.107197 166 5.0399882 180 7.02856624 18 WKEATT 1.35 10.7 80 3.9162301218 WKEATTT 1.22 6.2 108 6.03090101 120 .300033045 180 3.89999705 19KEATTT .71 6.7 80 3.87038548 19 KEATTTL .55 B 94 10.8301427 1153.54324115 166 6.61371042 20 EATTTL −.09 4.7 108 4.84178659 20 EATTTLF−.4 8.3 95 8.81271666 180 4.89999533 180 7.39999914 21 ATTTLF −1 3.4 802.02960213 21 ATTTLFC −1.26 8.7 80 5.55101217 112 1.81597681 1804.74285394 180 .599999107 22 TTTLFC −1.09 4.1 80 2.54250479 22 TTTLFCA−1.26 9.2 80 4.52553122 136 1.39839332 180 3.65714207 23 TTLFCA −1.111.4 80 3.3632097 23 TTLFCAS −1.25 1.1 80 5.52825776 157 .332518758 1305.40023235 24 TLFCAS −.99 3.5 80 3.32553457 24 TLFCASD −.85 8.4 803.87846076 147 1.80392438 119 7.0806631 180 5.94284743 25 LFCASD −.428.1 80 2.06205252 25 LFCASDA −1.21 11.6 80 4.08829296 122 4.25830797 1156.42577424 180 2.49999907 180 4.99999326 26 PCASDA −.21 6.8 803.58038047 26 PCASDAK −.11 12.7 94 5.27711073 139 4.84654054 1509.17980169 27 CASDAK .71 6.7 113 3.91223995 27 CASDAKA .04 3.8 603.77241193 180 6.69999644 123 7.36122555 180 10.9428509 28 ASDAKA .6 .680 2.28563281 28 ASDAKAY .58 9 80 3.54848783 157 6.39032513 15010.4603253 29 SDAKAY .5 2.6 80 3.2211604 29 SDAKAYS 95 2.2 60 .944789442136 6.65130434 180 11.6571397 30 DAKAYS .5 8 60 3.01974627 30 DAKAYST.94 2.8 80 .97233936 136 6.39531506 180 11.2571407 31 AKAYST −.07 4.4115 5.43526965 31 AKAYSTE .94 4.7 80 4.07367275 148 10.3211839 32 KAYSTE.51 2.7 80 6.27726053 32 KAYSTEV .6 3.4 80 6.27920693 144 6.09214551 14613.1855076 33 AYSTEV −.24 3.6 80 4.62499407 33 AYSTEVH −.03 1.49 805.29178076 180 6.99999752 139 9.29903238 34 YSTEVH .51 1.9 105 5.151836234 YSTEVNH .72 4 117 9.71864323 180 11.4999926 35 STEVHH .93 .2 80.963303906 35 STEVHNV −.06 6.2 128 12.8286227 180 9.99999872 36 TEVHNV.63 1.6 60 3.30736594 36 TEVHNVW −.05 .19 127 13.7231702 146 8.150286437 EVHNVW 1.26 3.4 134 10.0166439 37 EVHNVWA −.4 3.9 136 14.2693646 38VHNVWA .68 1.3 131 9.91647749 38 VHNVWAT −.81 1 94 6.01966306 14810.1310171 39 HNVWAT .86 .19 116 6.66449943 39 HNVWATH −.28 .4 884.30849285 180 11.8285674 40 NVWAT0 .86 1 123 6.65369771 40 NVWATHA −.461.8 84 5.69730511 161 10.5891462 41 VWATHA .75 1.69 133 9.64004539 41VWATHAC −1.32 .29 80 2.72499528 133 4.94525044 180 7.48570996 42 WATHAC.63 0 112 6.64659016 42 WATHACV −1.32 8.3 87 3.62934111 149 5.9109865943 ATHACV .01 6.1 80 6.1106703 43 ATHACVP −1.22 3.5 80 6.638677 1804.899999 146 6.75061693 44 THACVP .09 5.6 60 5.55283546 44 THACVPT −.86.29 80 6.79346425 180 5.39999651 135 5.22522487 45 HACVPT .1 4.4 605.17273346 45 HACVPTD −.46 10.6 80 5.70807306 180 5.39999715 1807.8571393 46 ACVPTD −.07 5.6 60 2.90366341 46 ACVPTDP −.16 14.3 803.93828552 160 4.39999676 180 6.14285612 47 CVPTDP .01 5.1 60 3.300853647 CVPTDPN .59 13.7 98 6.06728507 158 3.98100801 180 7.3999986 48 VPTDPN.21 5.1 80 3.65549613 46 VPTDPNP 1.18 6.6 102 5.52642539 180 5.09999822180 10.0857082 49 PTDPNP .46 2.4 88 3.38300557 49 PTDPNPQ 2.28 2.8 932.49422442 180 3.59999964 180 5.91428331 50 TDPNPQ 5 2.2 80 2.7184626850 TDPNPQE 2.55 2.8 91 2.95517847 170 3.80175964 180 5.65714194 51DPNPQE 1.06 0 80 4.14976691 51 DPNPQEV 1.85 5.8 90 7.87338593 1424.8530168 144 9.90699754 52 PNPQEV .31 1.5 143 4.58159443 52 PNPQEVV .7511.6 104 9.82802 180 1.05713855 53 NPQEVV .06 .4 100 4.89667866 53NPQEVVL −.02 20.8 80 6.77921655 147 6.7977L978 54 PQEVVL −.27 8 804.88563707 54 PQEVVLG −.46 16.2 80 10.712972 180 4.59999854 1806.25713895 80 4.22460786 55 QEVVLGN −.19 2.7 80 13.0618563 55 QEVVLG−.54 6.6 136 4.03179431 133 7.43431692 56 EVVLGN .54 3.2 80 4.7537475 56EVVLGNV −1.29 4.6 80 14.8577324 143 5.23077801 148 12.1496517 57 VVLGNV−.29 1.9 80 1.81938005 57 VVLGNVT −1.69 12.2 80 7.97331492 1321.94924385 146 9.18636992 58 VLGNVT 1.11 3.2 80 1.36359393 58 VLGNVTE−.59 7.6 98 14.3811965 180 2.59999936 59 LGNVTE −.36 4.3 115 6.0535825159 LGNVTEN .51 7.6 99 13.327365 180 1.89999906 60 GNVTEN −.02 5.7 803.07264158 60 CNVTENF .65 4.5 96 12.4977417 130 5.08870785 18011.4571327 61 NVTENF −.17 2.4 95 6.95264042 61 NVTENFN 1.1 4.2 10413.410322 180 .999997219 180 13.5999856 62 VTENFN −.17 3.2 94 6.8750269662 VTENFNM .32 1.2 110 10.3891878 180 .99999923 180 13.4285586 63 TENFNM−.14 6.4 80 4.31543897 63 TENFNMW 1.05 5.2 80 3.03805257 128 5.52726887145 9.71945235 64 ENFNMW .5 1.6 80 6.56133261 64 ENFNMWK 1.51 1.3 802.85710325 146 7.0171153 134 11.1247084 65 NFNMWK .5 7.2 80 4.8305368865 NFNMWKN 1.51 4.1 80 3.36279834 147 5.17803036 180 10.6857106 66FNMWKN .5 10.2 80 5.34455775 66 FNMWKNN 1.51 12.1 148 9.50267953 1475.54090488 67 NMWKNN .94 1.09 84 6.47598203 67 NMWKNNM 1.64 2.6 8010.3115109 147 8.85827898 68 MWKNNM .7 6 80 6.10042863 68 MWKNNMV .545.5 80 9.63372205 140 5.32945602 127 7.14594883 180 1.74284865 69 WKNNMV.66 9.2 80 2.50153618 69 WKNNMVE 1.31 10.9 80 11.0435161 120 4.27897212141 7.54448264 180 .599996358 70 KNNMVE .6 3.2 80 5.58419524 70 KNNMVEQ1.68 8.1 80 12.1178417 142 6.14937436 71 NNMVEQ .13 2.6 96 4.73472565 71NNMVEQM .95 0 90 14.9537026 72 NMVEQM −.12 4.5 100 6.00683252 72 NMVEQMH.28 3.7 96 14.1948524 180 2.31426043 73 MVEQMH .51 2.7 115 9.19182903 73MVEQMHE .28 3.7 97 14.1805622 180 4.1142807 74 VEQMHE 1.23 4 1168.62558323 74 VEQMHED 1.05 3.7 94 14.283656 149 5.84410789 75 EQMHED1.98 8.1 80 6.34436198 75 EQMHEDI 1.39 .1 91 12.326519 180 4.59999931 76QMHEDI .31 3.9 80 12.6132845 76 QMHEDII .64 1.2 90 12.1656749 18012.4999944 166 4.38843143 77 MHEDII −.89 16.7 80 14.797617 77 MHEDIIS.25 3.9 80 10.5667494 147 7.21621384 157 1.58692131 78 HEDIIS −.62 23.780 15.0471601 78 HEDIISL −.02 11.3 114 9.62047164 141 7.85709809 1805.08570813 79 EDIISL −1.59 7.5 81 15.6576936 79 EDIISLW .18 7.3 1169.13994576 100 2.09999726 180 5.31426206 80 DIISLW −1.52 12.9 8012.6258975 80 DIISLWD .18 .69 80 2.75431712 140 13.6672029 12311.6897921 180 1.31428269 81 IISLWD −1.52 18.3 80 4.8601113 81 IISLWDV.18 10.7 100 7.92396483 124 10.62247 180 5.31428532 82 ISLWDQ −.32 15.180 6.41076427 82 ISLWDQS 55 12.6 80 6.14412544 164 9.73681878 1807.05713941 83 SLWDQS .9 1.6 87 6.61515331 83 SLWDQSL .27 2.6 8012.1215222 147 7.59794494 84 LWDQSL .55 5.9 80 6.51625779 84 LWDQSLK .71.3 81 13.5587838 141 6.2593098 149 10.5987673 85 WDQSLK 1.35 5.1 805.92760332 85 WDQSLKP 1.46 6.2 89 9.49759941 151 5.29558905 1807.48S71071 86 DVSLKP .76 2.3 87 5.08496961 86 DQSLKPC 1 4.8 9712.4768136 180 7.89999554 87 QSLKPC .11 3.3 122 5.20035816 87 QSLKPCV−.1 5.6 95 14.484075 159 5.67278754 88 SLKPCV −.17 4 89 4.72437198 88SLKPCVK −.05 3.7 100 14.2863777 180 5.59999792 180 12.542844 89 LKPCVK.28 .7 97 7.84178318 89 LKPCVKL −.7 5.8 107 14.9608929 180 8.29999305180 14.799984 90 KPCVKL .28 2.3 99 7.08316366 90 KPCVKLT −.06 2.2 10711.1829906 180 8.29999385 180 12.4571346 91 PCVKLT −.29 3.3 1325.51747666 91 PCVKLTP .39 3.6 127 12.9245369 92 CVKLTP −.29 2.7 1295.47302639 92 CVKLTPL −1.16 1.3 80 .824544342 133 13.7445971 93 VKLTPL−.42 1.9 80 1.72145914 93 VKLTPLC −1.16 .6 80 2.17205276 138 6.86166129132 12.8299583 94 KLTPLC .34 3.6 124 5.85689593

Five peptides were selected from within residues 35 through 137 of thegp120 surface protein of HIV.

Peptide number (1, above) which spans blocks 1-5 (6 amino acids perblock) has delta values (termed ANGLE) consistent with a helicalstructure as predicted by both the Hopp/Woods computer program (blocklength of 6 amino acids) and the Kyte/Doolittle computer program (blocklength of 7 amino acids).

Peptide number (2, above) which spans blocks 23-28 has a peak ofdifferential hydrophilicity (a marked change in mean hydrophilicitybetween the first-half and second-half of a block of amino acids) thatis predicted by both programs.

Peptide number (3, above) which spans blocks 56-63 has delta valuesconsistent with a helical structure (Kyte/Doolittle) and a peak ofhydrophilicity (both programs).

Peptide number (4, above) which spans blocks 76-83 has a peak ofdifferential hydrophilicity (both programs).

Peptide number (5, above) which spans blocks 87-94 has delta valuesconsistent with helical structures (both programs).

All five of these peptides exhibit negative mean hydrophilicity valuesindicating that they are poor antibody binding sites.

Five other conserved regions of the two HIV envelope proteins can besimilarly analyzed and putative T cell-active peptides selected. Theseregions include residues 204-279 (C2 or conserved region 2), 415-458(C3), 470-510 (C4), 511-616 (C5) and 654-745 (C6) (Modrow et al., J.Virology, 61:570,1987).

Similar computer analysis of the gag gene of HIV has revealed several Tcell epitopes from within the core or gag gene of HIV (Coates et al.,Nature, 326:549, 1987). These peptides are shown below, with theirresidue position numbers in the protein shown above each peptide.

 56        62    EGCRQIL  74        85    ELRSLYNTVAT 170            180   VIPMFSALSEG 199       206    AMQMLKET 298       305    YVDREYKT333       342    KTILKALGPA 346       355    EMMTACQGV 367       375   AEAMSQVTN

Such synthetic peptides (either from the surface proteins or the coreproteins) are able to induce a cell-mediated response sufficient todestroy virus-infected cells bearing the corresponding HIV proteinepitopes on their cell surfaces, or as suggested by the work Walker etal., (Science, 234:1563-1566, 1986) inhibit the growth of the virus.

As an alternate approach to identify T cell active peptides, it may benecessary to thoroughly cover the protein sequence in question. In thiscase, overlapping 15-amino acid peptides (15 mers) can be made (thesecond peptide overlaps with the C-terminal 5 amino acids of the firstpeptide, the third overlaps the second, etc.) across the completeconserved amino acid sequence of both gp120 and gp41.

All of these peptides can be made, for example, by the solid phaseMerrifield-type synthesis but can also be made by liquid phase synthesisor recombinant DNA-related methods known to those skilled in therelevant arts. A further description of the basic solid phase synthesismethod, for example, can be found in the literature (i.e., M. Bodanskyet al., Peptide Synthesis, John Wiley and Sons, Second Edition, 1976, aswell as in other reference works known to those skilled in this type ofchemistry. The so-called “bag” technique described in Houghten, Proc.Natl. Acad. Sci. USA, 82:5131-5135 (1985) is also useful. Appropriateprotective groups usable in such synthesis and their abbreviations willbe found in the above reference, as well as in J. F. W. Mcomie,Protective Groups in organic Chemistry, Plenum Press, New York, 1973).

Several peptides were prepared using the before-described techniques.Illustrative peptides so prepared are discussed hereinafter.

Of those peptides that can be so prepared, an exemplary peptide usefulfor preparing a multimer as discussed hereinafter includes an amino acidresidue sequence whose formula corresponds to one of those shown below,from left to right and in the direction from amino-terminus tocarboxy-terminus:

—EQLWVTVYYGVPV—,

—VYYGVPVWKEA—,

—GVPVWKEATTLFC—,

—AHKVWATHACV—,

—CVPTNPVPQEVV—,

—VLENVTENFNM—,

—NNMVEQMHEDII—,

—EQMHEDIISLWDQ—,

—LWDQSLKPCVKLT—,

—SLKPCVKLTPLC—,

—SVITQACSKVSFE—,

—FEPIPIHYCAFPGF—,

—KKFNGTGPCTN—,

—GTGPCTNVSTVQC—,

—VQCTHGIRPVVSTQ—,

—YLRDQQLLGIWGC—,

—FLGFLGAAGSTMGAASLTLTVQARQ—,

—CRIKQIINMWQGVGKAMYA—,

—CRIKQIINMWQGVGKAMYAPPIGGQIRC—,

—EGCRQIL—,

—ELRSLYNTVAT—,

—VIPMFSALSEG—,

—AMQMLKET—,

—YVDREYKT—,

—KTILKALGPA—, and

—EMMTACQGV—.

In the list above, and elsewhere herein, hyphens at the amino- andcarboxy-termini of a sequence are intended to imply that one or moreadditional amino acid residues can be present in a peptide sequence, asdiscussed before.

Preferably, a useful peptide having a sequence shown hereinabove isutilized without additional residues at either terminus, except forcysteine and lysine residues as are discussed hereinafter. Such apeptide has a sequence, as discussed before, that corresponds to aformula shown below:

EQLWVTVYYGVPV,

VYYGVPVWKEA,

GVPVWKEATTLFC,

AHKVWATHACV,

CVPTNPVPQEVV,

VLENVTENFNM,

NNMVEQMHEDII,

EQMHEDIISLWDQ,

LWDQSLKPCVKLT,

SLKPCVKLTPLC,

SVITQACSKVSFE,

FEPIPIHYCAFPGF,

KKFNGTGPCTN,

GTGPCTNVSTVQC,

VQCTHGIRPVVSTQ,

YLRDQQLLGIWGC,

FLGFLGAAGSTMGAASLTLTVQARQ,

CRIKQIINMWQGVGKAMYA,

CRIKQIINMWQGVGKAMYAPPIGGQIRC,

EGCRQIL,

ELRSLYNTVAT,

VIPMFSALSEG,

AMQMLKET,

YVDREYKT,

KTILKALGPA, and

EMMTACQGV.

A preferred peptide includes a sequence, as discussed before, thatcorresponds to a formula shown below.

—LWDQSLKPCVKLT—,

—GVPVWKEATTLFC—,

—GTGPCTNVSTVQC—,

—YLRDQQLLGIWQC—,

—FLGFLGAAGSTMGAASLTLTQARQ—,

—CRIKQIINMWQGVGKAMYA—,

—EQLWVTVYYGVPV—,

—VYYGVPVWKEA—, and

—SVITQACSKVSFE—.

A particularly preferred peptide, except for the lysine and cysteineresidues discussed hereinafter, corresponds to a formula shown below.

LWDQSLKPCVKLT,

GVPVWKEATTLFC,

GTGPCTNVSTVQC,

YLRDQQLLGIWQC,

FLGFLGAAGSTMGAASLTLTQARQ,

CRIKQIINMWQGVGKAMYAPPIGGQIRC,

EQLWVTVYYGVPV,

VYYGVPVWKEA, and

SVITQACSKVSFE.

Some of the before-enumerated peptides have been disclosed in whole orin part by others as containing T cell epitopes. However, thosedisclosures did not teach or suggest the multimers that are discussedhereinafter.

For example, Berzofsky et al., Nature, 334:706-708 (1988) and Cease etal., Proc. Natl. Acad. Sci. USA, 84:4249-4253 (1987) disclosed twopeptides having the sequences, as shown before, that are represented bythe formulas

KQIINMWQGVGKAMYA, and

HEDIISLWDQSLK

that were said to stimulate T cells of mice immunized with the peptideor a recombinant molecule containing a large portion of the gp120molecules as well as in humans who had previously been immunized with arecombinant vaccinia virus that expressed the HIV gp160 protein.

Takahashi et al., Proc. Natl. Acad. Sci. USA, 85:3105-3109 (1988)prepared fifty-five peptides corresponding to much of the gp160 moleculeof HIV, and studied the T cell stimulatory effect of those peptides oncells from mice immunized with a recombinant vaccinia virus thatexpressed gp160. Those workers found a single peptide from the gp120sequence to be an immunodominant site for stimulation of cytotoxic Tlymphocytes, and that that peptide overlapped a B cell epitope capableof evoking virus-neutralizing antibody responses in both animals andhumans. That epitope was located at positions 308-322 of gp120 and wassaid by those workers to be a highly variable sequence among differentisolates of HIV.

Thus, being a B cell epitope and being highly variable in sequence, theimmunodominant peptide of Takahashi et al. has little bearing here. Fouradditional peptides (positions 343-357, 637-651, 657-671 and 780-794)were also said to appear to marginally sensitize target cells.

Of the preferred and particularly preferred peptides disclosedhereinbefore that are useful for preparation of the multimers discussedhereinafter, several are believed to be new, whereas others have beendisclosed in whole or in part of others. Those new peptides are mostpreferred and consist essentially of a sequence, written from left toright and in the direction from amino-terminus to carboxy-terminus,represented by a formula shown below:

YLRDQQLLGIWGC,

FLGFLGAAGSTMGAASLTLTQARQ,

EQLWVTVYYGVPV,

VYYGVPVWKEA,

SVITQACSKVSFE,

GVPVWKEATTLFC,

AHKVWATHACV,

CVPTNPVPQEVV,

SLKPCVKLTPLC,

FEPIPIHYCAFPGF,

EGCRQIL,

ELRSLYNTVAT,

VIPMFSALSEG,

AMQMLKET,

YVDREYKT,

KTILKALGPA, and

EMMTACQGV.

The above new peptides can also be included in a longer peptide having asequence of up to about 30 amino acid residues. Such a longer peptideconsists essentially of an amino acid residue sequence, from left toright and in the direction from amino-terminus to carboxy-terminus,represented by a formula shown below:

—YLRDQQLLGIWGC—,

—FLGFLGAAGSTMGAASLTLTQARQ—,

—EQLWVTVYYGVPV—,

—VYYGVPVWKEA—,

—SVITQACSKVSFE—,

—GVPVWKEATTLFC—,

—AHKVWATHACV—,

—CVPTNPVPQEVV—,

—SLKPCVKLTPLC—,

—FEPIPIHYCAFPGF—,

—EGCRQIL—,

—ELRSLYNTVAT—,

—VIPMFSALSEG—,

—AMQMLKT—,

—YVDREYKT—,

—KTILKALGPA—, and

—EMMTACQGV—.

The most preferred peptides of the group described immediately aboveconsist essentially of a sequence of up to about 30 amino acid residues,as shown before, represented by a formula shown below:

—YLRDQQLLGIWGC—,

—FLGAAGSTMGAASLTLTVARQ—,

—EQLWVTVYYGVPV—,

—VYYGVPVWKEA—,

—SVITQACSKVSFE—, and

—GVPVWKEATTLFC—,

C. The Multimer and Composition

A useful peptide is itself utilized in an aqueous composition orinoculum that contains dissolved or dispersed therein a multimeric formof the peptide. The peptide multimer is usually hereinafter referred toas being dispersed in water for greater ease of expression and since asolution can be viewed as the ultimate form of a dispersion.

These peptides elicit a T cell response but not a substantial antibodyresponse, when introduced into an immunocompetent host animal, (amammal) such as a laboratory mouse or rat, a goat, an ape such aschimpanzee or a human. Therefore, when suitably prepared, a peptidemultimer composition of the present invention stimulates T cell immunity(e.g., cytotoxic T cells) without producing a substantial humoralantibody response. The peptide multimer composition of the presentinvention primes T cells in a way that, when the infecting virus appearsat a later date, memory T cells are activated to result in acell-mediated immune response that destroys target cells that have thecorresponding HIV protein or a portion thereof on their cell surfaces,and thereby the virus.

The activation of only T cells without an antibody response is importantbecause it is believed that antibodies to most regions of the viralenvelope protein may stimulate the infectivity of the virus. This latterpoint renders most viral surface envelope antigen preparations (e.g.,intact gp120 and gp41 that contain both B- and T-cell epitopes)ineffective as vaccines. Barnes, Science, 236:255, (1987). The Barnesarticle reported that about 20 chimpanzees had been given variousprototype vaccines (containing B- and T-cell epitopes) and some werechallenged by injecting virus, but the results indicated that none ofthe vaccines prevented infection by infectious HIV. In contrast, thisinvention provides a suitable T cell response that produces cytotoxic Tcells or other types of T cell responses that kill or otherwiseneutralize target cells such as T lymphocytes that express an HIVprotein or a portion of an HIV protein on the target cell surface.

It should be emphasized that an effective peptide multimer can in somecases induce a low to moderate level antibody response and still beuseful in an effective composition. In this case, the inducedanti-peptide antibodies are incapable of recognizing or detecting themature native protein such as gp160 to which the peptide of the multimercorresponds in sequence. Thus, the anti-peptide antibodies induced bythe T cell active peptide must not be substantially capable of bindingto the intact, infectious virus. It is well known that anti-peptideantibodies to certain regions of a given protein may not recognize thenative protein (for example, see the work of Ho et al., J. Virol.,61:2024, 1987).

The use of synthetic peptides that are T cell active but that are notimmunogenic for native virus (anti-peptide antibodies that are unable todetect or immunoreact with the virus particle) can have an addedadvantage in that inherent immunological memory should be superior forpeptide vaccines of the present invention.

The composition or inoculum contains a before-described peptide in amultimeric form. Exemplary of such multimers are surfactant-likemicelles and polymers, examples of each of which are discussedhereinafter.

In one type of multimer synthesis, the N-terminal end of each peptide islinked to a di-C₁₂-C₁₈ fatty acid amide of a lysine-terminated peptidespacer such as a dipalmityl-lysyl-glycyl-glycyl sequence to serve as acarrier as described by T. P. Hopp (Mol. Immunol., 21:13, 1984). Otheruseful C₁₂-C₁₈ fatty acids include lauric, myristic, stearic, oleic andpalmitoleic acids.

An example of this type of structure is shown below:

In addition to the amino-terminal lysyl residue, the spacer peptide cancontain one to about five additional residues. Substantially any aminoacid residue can be utilized so long as it does not interfere with the Tcell immunizing capacity of an aqueous composition containing themultimer or with the capacity of the di-amide reaction product to formsurfacetant-like micelles in an aqueous composition. One to about threeglycyl residues per spacer peptide are preferred.

The before-described peptide and the amino-terminal lysylresidue-containing peptide spacer are peptide-bonded together, and canthus be viewed as a composite polypeptide. The useful diamide is thus areaction product of the alpha- and epsilon-amino groups of theamino-terminal lysyl residue and two moles per composite polypeptide ofthe C₁₂-C₁₈ fatty acid. The composite polypeptide can thus be preparedas a single sequence and amidified before or after removal from theresin, where solid phase synthesis is used, by conventional techniques.

The phrase “surfactant-like micelle” is used herein to emphasize that,in an aqueous composition, the di-amidolysyl composite polypeptideappears to form micelles similar to those formed by surfactants and todistinguish such multimers from submicroscopic structural units ofprotoplasm built up from polymeric molecules that are also sometimesreferred to as micelles. The word “micelle” is also sometimes usedherein, and when so used has the same meaning as surfactant-likemicelle.

Another multimer form of a previously described peptide is a polymerhaving a plurality of peptide repeating units. In this case, a peptidecontaining two terminal cysteines as part of its natural sequence can beselected and synthesized. A peptide lacking such cysteines can bemodified by the addition of one or two extra cysteines to the N- andC-terminal ends, respectively. The presence of two cysteines per peptidepermits polymerization of the subunit peptide by air oxidation to formoxidized cysteine(cystine)-linked polymers and/or cyclic peptides. Suchmultimers enhance immune recognition of the peptide without the need ofa carrier.

An example of this type of structure is shown below:

The lysine-terminated spacer peptide can contain one to about five aminoacid residues in addition to the lysyl residue, and the one or two addedterminal cysteine residues are not included in counting the length of apeptide of the present invention. A peptide containing terminal cysteineresidues is referred to as a di-cysteine-terminated peptide or moresimply, a di-Cys peptide. Details for preparing polymers containingdi-Cys peptide repeating units are provided hereinafter.

It should also be noted that a peptide multimer of a composition cancontain more than one, active, T cell stimulating peptide as describedpreviously. The inclusion of more than one such active peptide permitsactivation by more than a single T cell epitope to a single HIV protein,as well as to a plurality of HIV proteins. Such inclusion of peptides ofdifferent sequences can also avoid non-response in the host animal thatis immunized. In addition, a multimer can also include an inactivepeptide; i.e., a peptide that does not induce T cell activation orantibodies that immunoreact with a native HIV protein, to enhance waterdispersibility, for example.

An aqueous composition (inoculum) of the present invention comprises animmunologically effective amount of a before-described peptide multimerdissolved or dispersed in a pharmaceutically acceptable aqueous medium.Such compositions are also referred to as inocula, as noted before.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that do not produce an allergic or similar untowardreaction when administered to a human.

The preparation of an aqueous composition that contains an immunizingmolecule such as a before-described peptide multimer as an activeingredient is well understood in the art. Typically, such compositionsare prepared as injectables, either as liquid solutions or suspensions;solid forms suitable for solution in, or suspension in, liquid prior toinjection can also be prepared. The preparation can also be emulsified.

The active immunogenic peptide multimer is dissolved or dispersed in anexcipient that is pharmaceutically acceptable and compatible with theactive T cell immunogen as is well known. Suitable excipients are, forexample, water, saline, phosphate buffered saline (PBS), dextrose,glycerol, ethanol, or the like and combinations thereof. In addition, ifdesired, the composition can contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents oradjuvants that enhance the effectiveness of the vaccine.

The composition is conventionally administered (introduced)parenterally, by injection, for example, intraperitoneally,intravenously, intradermally, subcutaneously or intramuscularly.Additional formulations that are suitable for other modes ofadministration include suppositories and, in some cases, oralformulations. For suppositories, traditional binders and carriers caninclude, for example, polyalkalene glycols or triglycerides; suchsuppositories can be formed from mixtures containing the activeingredient in the range of 0.5 percent to 10 percent, preferably 1-2percent. Oral formulations include such normally employed excipients as,for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonateand the like.

A peptide multimer can be formulated into a composition in a neutral orsalt form. Pharmaceutically acceptable salts, include the acid additionsalts (formed with the free amino groups of the peptide) and which areformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine, and thelike.

Similarly, peptides and peptide multimers of this invention can formsalts with fluosilicic acid. These salts are useful as mothproofingagents in accordance with the teachings of U.S. Pat. No. 1,915,334 andU.S. Pat. No. 2,075,359. The instant peptides and peptide multimers alsoform salts with thiocyanic acid which, in turn, can be condensed withformaldehyde to form resinous materials useful as pickling inhibitors inaccordance with U.S. Pat. No. 2,425,320 and U.S. Pat. No. 2,606,155.Salts of the peptide and peptide multimers with trichloroacetic acid areuseful as herbicides against Johnson grass, yellow foxtail, Bermudagrass, quack grass, and the like. Salts formed between ammonia and acarboxylic acid present in the peptides and peptide multimers of thisinvention can be used as a source of nitrogen for leguminous plants suchas peas.

A composition is administered in a manner compatible with the dosageformulation, and in such amount as is immunologically effective. By“immunologically effective amount” is meant an amount of composition isused that contains an amount of a peptide multimer sufficient to inducecellular immunity in the host animal (mammal) such as by the inductionof anti-HIV cytotoxic T cells. The presence of such cytotoxic T cells isassayed as discussed hereinafter.

The quantity of multimer peptide and volume of composition to beadministered depends on the host animal to be immunized, the capacity ofthe host animal's immune system to activate T cells, and the degree ofprotection desired. Precise amounts of active peptide multimer requiredto be administered depend on the judgment of the practitioner and arepeculiar to each individual. However, suitable dosage ranges are of theorder of about 10 micrograms (μg) to about 500 milligrams, preferablyabout 50 μg to about 1 mg, and more preferably about 100 micrograms ofactive ingredient peptide multimer per individual. A minimal volume of acomposition required to disperse the immunizing amount of peptidemultimer is typically utilized. Suitable regimes for initialadministration and booster shots are also variable, but are typified byan initial administration followed in one or two week intervals by asubsequent injection or other administration.

A composition can also include an adjuvant as part of the excipient.Adjuvants such as complete Freund's adjuvant (CFA), incomplete Freund'sadjuvant (IFA) for use in laboratory host mammals are well known in theart, and are used illustratively herein. Pharmaceutically acceptableadjuvants such as alum can also be used.

Typical mammals (host animals) used in practicing a method of thisinvention include mice, rabbits, goats, primates, humans and the like.

In a usual screening procedure, each peptide multimer preparation isfirst assayed in mice, for example, to screen for an appropriate T cellactive peptide multimer. T cell active peptide multimers are assayed byinjecting a before-described composition into mice, and then testing Tcells recovered from the murine lymph nodes one to three weeks afterinoculation with the peptide multimer-containing composition. Themeasurement of activation or priming of T cells is done by T cellproliferation tests and/or interleukin-2 production (Milich et al., J.Exp. Med., 164:532, 1986).

Two types of T cell active peptides should be found. The more prevalentgroup of peptides prime (activate) T cells that respond in test tubeassays to only the peptide and not the corresponding native HIV surfaceprotein. The second group of peptides prime T cells to respond to boththe peptide and the native HIV protein. It is this latter group ofpeptides that induce protective immunity in the immunized host. Aplurality of strains of mice that vary in their histocompatibility genesare used for these screenings. Peptides that have a broad response inthe various MHC genotypes are selected for further study in primates,and finally humans. Exemplary assay procedures are found hereinafter.

T cell active peptide multimers are also screened to identify thosepeptide multimers that lack B cell stimulatory activity. This isaccomplished by injecting each peptide multimer into smallimmunocompetent animals (various strains of mice) to identify thosepeptides that fail to generate an antibody response to the native HIVprotein to whose sequence the peptides correspond in part such as thegp120, gp41 or core proteins, for example. These animals should notproduce anti-peptide antibodies that bind to (immunoreact with) thecorresponding native viral protein. Those selected peptides that induceT cell activation, but do not induce an antibody response to theircorrelative or corresponding native protein are then assayed in baboonsor other apes and monitored to confirm the lack of anti-peptide antibodyproduction in baboon sera.

At this stage, mixtures of peptides are preferably employed in themultimer to provide a broad spectrum of coverage usually needed for aneffective T cell activating composition. Peptide mixtures are thenincorporated into a multimer-containing composition and assayed in asuitable animal that allows replication of the AIDS virus (e.g.,chimpanzees) to test for priming of T cells. Peptides that are moreactive are used to immunize chimpanzees in a virus challenge study. Asuccessful protection study prevents viremia without eliciting asignificant humoral antibody response, but primes T cells for in vitroresponses to the envelope antigens. The virus is then neutralized bycell mediated immunity.

A composition containing a peptide multimer in an immunologicallyeffective amount is thus obtained that can induce a killing effectiveamount of cytotoxic T cells. Those cytotoxic T cells are capable ofkilling target cells, when contacted in vitro or in vivo with suchtarget cells like T lymphocytes or other cells such as P815 mouse cells(when the cytotoxic T cells are from a mouse; P815 mouse cells areavailable from Dr. Fernando Plata of the Pasteur Institute, Paris,France) that exhibit a corresponding HIV protein or a portion of such aprotein on their cell surfaces.

As described before, it is not necessary to select a peptide thatcompletely lacks the capability to raise anti-peptide antibodies. Wheresuch antibodies are induced, the anti-peptide antibodies must not becapable of recognizing (immunoreacting with) the native envelopeproteins as measured, for example, either by immunoblotting proceduresor by other immunoabsorbent (ELISA) tests. What is important in thisparticular response is that anti-peptide antibodies against a certainpeptide sequence must not induce antibodies that bind to the infectiousvirus. Thus, in this case, T cell active peptides that raise low ormoderate levels of anti-peptide antibodies are screened to identifythose that fail to detect either intact virus preparations or viralsurface proteins by immunoabsorbent tests (ELISA) and/or immunoblotprocedures.

D. Methods

Methods constitute yet another aspect of the present invention.

A first method comprises the induction of T cell immunity to apreselected native HIV protein in a host animal such as a laboratoryanimal or a human as noted previously. Here an immunizing effectiveamount of a before-discussed active peptide multimer-containingcomposition is introduced into the host animal, and the host animal ismaintained for a time period sufficient for the T cell immunity todevelop. This immunization does not induce substantial production ofantibodies that immunoreact with the preselected HIV protein. Thisimmunization can be repeated or boosted from time to time as desired.

The preselected HIV native protein is a protein to which an activepeptide of the multimer corresponds in sequence. As already noted, anactive peptide multimer can include active peptides corresponding todifferent HIV proteins, and as a consequence, the above method can beused to induce T cell immunity to more than one HIV protein.

T cells from the immunized host animal can be collected and assayed fortheir having immunity to the preselected HIV protein using an assay suchas the proliferation assay discussed hereinafter.

The immunized T cells prepared as discussed in the above method can beutilized in a method of killing target cells that exhibit an HIV proteinor a portion thereof on their cell surfaces. In this method, such targetcells are contacted with a killing effective amount of cytotoxic T cellsthat have been immunized with a before-discussed composition, as alreadydiscussed. That contact is maintained for a time period sufficient tokill the target cells.

The above method can be carried out in vitro or in vivo. For in vitrostudies, immunized T cells are obtained from an immunized host animaland are admixed and contacted with the target cells in an appropriateaqueous medium such as RPMI medium. The admixture is thereafter assayedfor lysis of the target cells as by the ⁵¹Cr assay discussedhereinafter.

The target cells utilized can be HIV-infected cells that express andthereby exhibit HIV proteins such as core, gp120 and gp41 on their cellsurfaces. The target cells can also be cells such as spenocytes thatupon admixture with a peptide used in the multimer binds that peptide toits surface and thereby exhibits a portion of an HIV protein on itssurface. Further target cells include P815 mouse mastocytoma cells (ATCCTIB64 that have been further transferred to express HIV proteins, andare available from Dr. Fernando Plata of the Pasteur Institute, Paris,France, as noted before.

In an in vivo aspect of the above method, the target cells and immunizedcytotoxic T cells are supplied by the immunized host animal; i.e., thehost animal is infected with HIV, and HIV proteins or portions thereofare expressed on the surfaces of host cells such as T4⁺ cells. Once ahost animal such as a chimpanzee or human expresses HIV proteins on cellsurfaces, the animal is usually also viremic. As a consequence, adecrease in viremia of an infected host after immunization or theabsence of viremia after immunization and infection provide assays forthe above method.

In still another aspect of the above method, autologous or appropriatelymatched heterologous cytotoxic T cells are used. For autologous cells,immunization as discussed previously can be sufficient. However,immunized T cells can be recovered as already discussed, culturedfurther in the presence of an immunizing peptide multimer to proliferatethe cells, and then those proliferated cells can be re-introduced intothe same host animal to augment the effect obtained by immunizationalone.

For heterologous cells, a donor is immunized with a previously discussedcomposition and the donor's immunized T cells collected. Immunized Tcells from an appropriately matched donor, e.g. a syngeneic donor, canbe then introduced into an HIV-infected recipient as a passiveimmunization. Prior to the passive immunization, the matched donor cellscan be proliferated as discussed above and then utilized.

The maintenance time of contact between the target cells and effectorcytotoxic T cells can vary from about an hour to days, depending onseveral parameters, most importantly being whether the method is carriedout in vivo or in vitro. For in vivo methods, the maintenance time isthe lifetime of the cytotoxic T cells, which can be days to weeks. Forin vitro uses, maintenance times of one to about 10 hours, andpreferably about 2 to about 5 hours are generally used.

An important issue in considering the effectiveness of a peptidemultimer or method of this invention is whether the cell-mediated immunesystem can function in a previously immunized individual when at a latertime the immunized host animal is exposed to HIV which is infecting andaltering the function of T4 helper cells. The research findings ofBuller et al. (Nature, 328: 77, 1987) provide evidence that isconsistent with the hypothesis that a T cell active peptide can invoke acell mediated response in the absence of T4 helper cells. Their workdemonstrates that cytotoxic T cell responses can be induced in mice inthe absence of T helper cells; the end result was that mice beingstudied recovered from a viral disease without T helper cells.

Therapy for HIV-infected host animals such as people is alsocomtemplated by the present invention. A composition of this inventioncan thus be used to treat animal hosts that are already infected withHIV.

In this particular situation, it is important to consider that thetarget for cell mediated immunity includes not only the virus but moreimportantly the virus-infected cell. Such infected cells have not onlyviral envelope proteins on their surfaces but possibly glycosylated coreproteins (gag gene products) or their higher molecular weight precursorsas well (Naso et al., J. Virol., 45:1200, 1983). Therefore, T cellactive peptides from the gag gene of HIV as noted before are alsoselected, assayed and used for their affects on virus infected cells, asdiscussed above.

The T helper cell-independent cytotoxic T cell response, described byBuller et al., bodes well for the use of T cell active peptide multimersin the therapy of AIDS. Such a peptide multimer or a multimer containingmixture of peptides can mount an effective cell-mediated immune responseat a time when T4 cells are being infected and killed by the HIV. SinceT8 cells are resistant to HIV infection, a peptide multimer can activateand prime T8 cytotoxic cells permitting a specific virus-killingresponse in the AIDS patient even though the virus may be infecting andaltering the immune helper function of T4 cells.

Studies of Walker et al., (Nature, 328: 345, 1987) have demonstrated thepresence HIV-specific cytotoxic T cells in persons infected with HIV.These cytotoxic T cells were able to kill HIV antigen-containing Blymphocytes derived from the same patient in laboratory tests. Theirstudy showed that a monoclonal antibody specific for cytotoxic T cellswas able to inhibit the cell killing activity. These results support theimmunization approach described herein, and may have importantimplications for the use of T-cell active peptides and their multimersin the treatment of AIDS patients.

BEST MODE FOR CARRYING OUT THE INVENTION EXAMPLE 1—Preparation ofPeptides, Peptide Polymers and Peptide Micelles

Synthetic peptides of 7 to about 30 amino acid residues in length wereprepared corresponding to the selected conserved domains of the core andgp160 (gp120 and gp41) molecules using the solid-phase technique ofMerrifield described in J. Am. Chem. Soc. 85:2149-2154 (1963) using amodified Vega 250 automated peptide synthesizer or by the “bag” methoddescribed in Houghten, Proc. Natl. Acad. Sci, USA, 82:5131-5135 (1985).The t-butyloxycarbonyl (t-BOc) amino acid blocking groups and thehydrolysis of the peptide from the resin were carried out byhydrofluoric acid (HF) treatment at about zero degrees C for one hour.The peptide-containing mixture was then extracted with diethyl ether toremove non-peptide organic compounds and the synthesized peptides wereextracted from the resin with acetic acid (25 percent w/v).

Nineteen (19) synthetic peptides have been prepared that correspond toconserved domains of the gp120 molecule and the gp41 molecule by thisprocedure, and are listed in TABLE 2. The synthesized peptidescorrespond to designated conserved domains (regions) of gp160 in HIV asshown.

TABLE 2 AMINO ACID SEQUENCE OF SYNTHETIC PEPTIDES PEPTIDE LOCATION IN #AMINO ACID SEOUENCE¹ HIV ENVELOPE² 103 ³⁹EQLWVTVYYGVPV⁵¹ GP160-CR-1 104⁴⁵VYYGVPVWKEA⁵⁵ GP160-CR-1 105 ⁴⁸GVPVWKEATTLFC⁶¹ GP160-CR-1 106⁷²AHKVWATHACV⁸² GP160-CR-1 107 ⁸¹CVPTNPVPQEVV⁹² GP160-CR-1 108⁹²VLENVTENFNM¹⁰² GP160-CR-1 109 ¹⁰⁵NNMVEQMHEDII¹¹⁶ GP160-CR-1 110¹⁰⁹EQMHEDIISLWDQ¹²¹ GP160-CR-1 111 ¹¹⁸LWDQSLKPCVKLT¹³⁰ GP160-CR-1 112¹²¹SLKPCVKLTPLC¹³³ GP160-CR-1 113 ²⁰⁴SVITQACSKVSFE²¹⁶ GP160-CR-2 114²¹⁵FEPIPIHYCAFPGF²²⁸ GP160-CR-2 115 ²³⁶KKFNGTGPCTN²⁴⁶ GP160-CR-2 116²⁴⁰GTGPCTNVSTVQC²⁵² GP160-CR-2 117 ²⁵⁰VQCTHGIRPVVSTQ²⁶³ GP160-CR-2  61⁵⁸⁶YLRDQQLLGIWGC⁵⁹⁸ GP160-CR-5  63 ⁵¹⁹FLGFLGAAGSTMGAASLTLTVQ- GP160-CR-5ARQ⁵⁴³  65 ⁴¹⁷CRIKQIINMWQGVGKAMYA⁴³⁵ GP160-CR-3  67⁴¹⁷CRIKQIINMWQGVGKAMYAPP- GP160-CR-3 IGGQIRC⁴⁴⁴ ¹The N- and C-terminalamino acid residues of each peptide are numbered as to their position inthe gp160 amino acid residue sequence according to Modrow et al. Virol.,61:570 (1987). A dash (-) indicates that the sequence continues on thenext line. ²CR = Conserved Region

Two types of high molecular weight (multimeric) forms of the peptideslisted in TABLE 2 were prepared. The principal form of multimer was adi-cysteine (di-Cys terminated) polymer in which a plurality of peptideswere linked end-to-end by disulfide bonds. These di-cysteine polymerswere produced by adding a cysteine residue to the termini of eachpeptide during synthesis. The di-cysteine-terminated (di-Cys) peptideswere then dissolved (10 mg/ml) in ammonium bicarbonate (0.1M) at roomtemperature (about 25 degrees C.) and stirred for about 16 hours toeffect oxidation of the sulfhydryl groups to produce polymer forms ofthe peptides.

The second type of high molecular weight form produced was asurfactant-like micelle formed by linkage of an amino-terminallysine-containing spacer peptide (Lys-Gly-Gly-) to the peptide sequenceto form a composite polypeptide, and then coupling a C₁₂-C₁₈ fatty acid,such as palmitic acid, to both the alpha and epsilon amino groups by themethod described in Hopp, Mol. Immunol. 21:13-16 (1984), which isincorporated herein by reference. The C₁₂-C₁₈ fatty acid-containingpeptides produced are then extracted in acetic acid (95 percent), andutilized to form large micelles in the aqueous composition that exhibitincreased immunogenicity relative to the peptides.

Di-Cys polymer multimers of all of the peptides listed in TABLE 2 wereprepared. Aqueous peptide micelle multimers have been prepared ofpeptides designated 61, 63, 65 and 67, and are designated as peptides62, 64, 66 and 68, respectively. Peptides designated 103 through 117were utilized only in their di-Cys polymer multimeric forms.

The high molecular weight, multimeric forms produced correspond tomultiple copies of specific regions of gp120 and gp41 in HIV. For easeof designation, the multimer forms will be designated by the peptidenumber from which it is composed—that is, peptide 61 refers to thedi-Cys multimeric (polymeric) form of peptide 61 and peptide 66 refersto the aqueous micelle form of peptide 65, whereas peptide 103-117refers to a polymeric multimer.

Peptides 65 and 66 correspond to the region of gp120 that binds to thecell surface T₄ receptor. Peptides 63 and 64 correspond to a region nearthe amino-terminal portion of gp41 that represents a majorimmunodominant epitope seen by AIDS patients' serum.

EXAMPLE 2—Anti-Peptide Antibody Response

Aqueous compositions of the multimers; i.e., the di-Cys peptide polymersand micelles produced in EXAMPLE 1 were assayed for their ability, orlack of ability to elicit an anti-peptide antibody response in BALB/cmice, an immunocompetent mouse strain.

Groups of BALB/c mice (6-8-week-old females, 3 to 5 mice/group, CharlesRiver Laboratories) were immunized by subcutaneous (s.c.) orintraperitoneal (i.p.) injection of a peptide multimer (100μg/injection) in complete Freund's adjuvant (CFA) (1:1 ratio). Boosterinjections (50 μg of peptide multimer) in incomplete Freund's adjuvant(IFA) (1:1) were given at 6 and 10 weeks after the initial immunization.Each mouse was bled from its retro-orbital plexus at two-week intervalsand the serum was pooled for individual mice in each group.

An ELISA assay was performed on each serum to detect the presence ofanti-peptide antibodies utilizing peroxidase-conjugated goat anti-mouseIgG (obtained from Boehringer Mannheim Biochemicals, Indianapolis, Ind.)as the second antibody). Preliminary results for peptides 61-68 areshown in TABLE 3, whereas further refined results for peptides 61, 63,65, 67 and 103-117 are shown in TABLE 4.

It was found that the high molecular weight forms of peptides 65, 66,67, 68, 105 to 110, 112, 114, 115 and 117 elicited high antibody titers,whereas peptides 61, 62, 63, 64, 103, 104, 111, 113 and 116 producedvery low to negligible amounts of anti-peptide antibodies. Similarresults were obtained for antibody responses in B6C3 F1 mice (CharlesRiver Laboratories), another immunocompetent strain.

Some of the sera were further assayed for antibody response (reactivity)with native gp160, and the results, shown in TABLE 5, demonstrate thatthese peptides do not represent B cell epitopes since there was noimmunoreaction with native gp160.

TABLE 3 ANTIBODY RESPONSE OF VARIOUS PEPTIDES IN BALB/C MICE ELISA Titerin Bleed # Peptide Pre- # Immune 1 2 3 4 5 6 7 8 61 1:40 1:400 1:1001:100 1:100 1:100 1:400 1:200 1:400 62 1:20 1:100 1:100 1:100 1:1001:100 1:200 1:100 1:100 63 1:40 1:80 1:20 1:40 1:320 1:60 1:80 1:3201:5120 64 1:20 1:40 1:40 1:00 1:40 1:10 1:40 1:40 1:40 65 1:40 1:601:600 1:1 × 10⁴ 1:5 × 10⁴ 1:5 × 10⁴ 1:2 × 10⁵ 1:2 × 10⁵ 1:2 × 10⁵ 661:10 1:160 1:6 × 10³ 1:1 × 10⁵ 1:1 × 10⁵ 1:1 × 10⁵ 1:2 × 10⁴ 1:5 × 10⁴1:5 × 10⁴ 67 1:40 1:160 1:3 × 10³ 1:2 × 10⁴ 1:2 × 10⁴ 1:1 × 10⁵ 1:1 ×10⁵ 1:1 × 10⁵ 1:2 × 10⁵ 68 1:80 1:1600 1:1 × 10⁴ 1:1 × 10⁴ 1:1 × 10⁵ 1:1× 10⁵ 1:4 × 10⁵ 1:4 × 10⁵ 1:4 × 10⁵

TABLE 4 ANTIBODY RESPONSE OF VARIOUS PEPTIDES IN BALB/C MICE PEPTIDEELISA TITER  #61 AA 586-598 1:400  #63 AA 519-543 1:5120  #65 AA 417-4351:2 × 10⁵  #67 AA 417-444 1:8 × 10⁵ #103 AA 39-51 1:640 #104 AA 45-551:2000 #105 AA 48-61 1:5000 #106 AA 72-82 1:4 × 10⁵ #107 AA 81-92 1:1 ×10⁵ #108 AA 92-102 1:1 × 10⁵ #109 AA 105-116 1:8 × 10⁵ #110 AA 109-1211:6 × 10⁶ #111 AA 118-130 1:80 #112 AA 121-133 1:1 × 10⁵ #113 AA 204-2161:640 #114 AA 215-228 1:1 × 10⁶ #115 AA 236-246 1:4 × 10⁵ #116 AA240-252 1:640 #117 AA 250-263 1:8 × 10⁶

TABLE 5 T AND B CELL RESPONSES IN MICE TO HIV ENVELOPE GP160 DERIVEDSYNTHETIC PEPTIDE IMMUNOGENS In Vitro Proliferation of PLN Cells from*Antipeptide Antibody B₆C₃F₁ A · SW × Balb/c F₁ Reactivity to** PeptideAnalogous Analgous Analogous Immunogen Peptide GP 160 Peptide GP 160Peptide GP 160  61 ++ + ++ ++ − −  63 ++ ++ ++ ++ _+ −  65 ++ + ++++ ++++ −  67 ++ − ++++ ++ +++ − 103 + + +++ + − − 104 ++++ ++ +++ + ± − 105++++ +++ + − ± − 106 +++ + ++++ + ++ − 107 ++ ++ + + + − 108 + + + − + −109 ++ ± ± + +++ − 110 ++ − ++ + ++++ − 111 + ++ ± − − − 112 + + + + + −113 ++ + ++ + − − 114 ++ − ++ + ++++ − 115 ++ + ND ND ++ − 116 ++ − NDND − − 117 +++ − ND ND ++++ − *cpm values are corrected and categorizedaccording to unrelated antigen response in vitro. **Antibody raised inBalb/C mice, reactivity measured by ELISA and categorize according tothe end point. Not determined.

EXAMPLE 3—T Cell Responses

The high molecular weight, multimeric di-Cys peptide polymeric forms ofthe peptides described in EXAMPLE 1 were assayed for their elicitationof a T cell proliferative response by the method described in Millich etal., J. Immunol. 134:4194-4203 (1985), incorporated herein by reference.

Mice (3 or 5 mice/group) were injected in the right hind footpad with apeptide polymer (100 μg/injection) in complete Freund's adjuvant (1:1).Peptides 61, 63, 65 and 67 were injected into B6C3 F1 mice (H-2^(kxb)(Charles River Laboratories) and A.SWxBALB/C F1 mice (H-2^(sxd))(Jackson Labs, Bar Harbor, Me.). Draining popliteal lymph node (PLN)cells were harvested after ten (10) days, and cultured (2×10⁵cells/well) in 96-well microtiter plates in 0.2 ml of Click's medium(Click et al., Cellular Immunol. 3:264-276 (1972)] containing variousconcentrations of synthetic peptide, gp160, an unrelated proteinaceousmaterial or medium alone, for 96 hours at 37 degrees C. in a humidifiedatmosphere of 5 percent CO₂. During the final 16-18 hours of culturing,³H-thymidine (³H-TdR) (1 μCi/well, 6-7 Ci/mmole, ICN Radiochemicals) wasadded. The cells were harvested onto filter strips and ³H-TDRincorporation was monitored. The data are presented in FIGS. 1, 2, 3, 4and 5.

FIG. 1 illustrates the results for peptides 61, 63, 65, 67 in BALB/cmice, and those results are expressed as a stimulation index (SI)representing the fold increase in radioactivity counts in the presenceof antigen compared to background values where no antigen was added. TheSI values with the different peptides were compared to that obtainedwith tuberculin purified protein derivative (PPD) as a positive controlantigen.

FIGS. 2-5 illustrate the peptide-specific ³H-TdR incorporation for Tcell responses (delta cpm) in mice with differing majorhistocompatibility (MHC) haplotypes, B6C3 F1 (C57B1/6xC³H/HcJ) mice(FIGS. 2 and 4) and (A.SWxBALB/c) F1 mice (FIGS. 3 and 5), for all ofthe synthetic peptides. The ³H-TdR incorporation values represent thedifference between the radioactivity values obtained in wells containingantigen and in control wells without added antigen. The non-specificproliferation of PLN cells was determined by including an unrelatedpeptide in the assays, shown as a horizontal bar for each peptide.

All of the assayed peptides exhibited good T cell proliferativeresponses in B6C3 F1 mice, whereas all of the assayed peptides, exceptpeptides 105, 107, 109 and 111, exhibited good T cell proliferativeresponses in SWxBALB/c F1 mice.

It was demonstrated by the results above and those described in EXAMPLE2 that peptides 61, 63, 103, 104 and 113 do not stimulate anti-peptideantibody production but are very good immunogens, in their disulfide(di-Cys) polymeric form, for eliciting a strong T cell response directedagainst both the corresponding peptide and the native HIV envelopeprotein gp160.

T cell proliferation measured by ³H-TdR incorporation, was alsosimilarly assayed as a function of the T cell antigen concentration,using various amounts of native gp120 or gp160 as one control, and PPDas another control. PLN from B6C3 F1 mice were used in these studies.The results for peptides 104 and 105 versus gp120 are shown in FIGS. 6Aand 6B, respectively; those for peptides 61 and 63 versus gp160 areshown in FIGS. 7A and 7B, respectively; and those for peptides 65 and111 versus gp120 are shown in FIGS. 8A and 8B, respectively.

EXAMPLE 4—Induction of HIV-Specific Cytotoxic T Lymphocytes

Groups of 3 to 5 syngeneic female mice (6 to 8 weeks of age) areimmunized by injection in an appropriate site with an aqueouscomposition containing an immunizing (cytotoxic T cell stimulating)amount of either of the before-discussed multimers, in a mixture withCFA (1:1). Ten (10) days after immunization, draining PLN cells andspleen lymphocytes are obtained and restimulated in vitro by culturingfor six (6) days with the same synthetic peptide as immunogen.

The presence of cytotoxic T lymphocytes (CTL) is determined by a 6-hour⁵¹Cr release assay as follows. The PLN and spleen cells are maintainedfor six days at 37 degrees C. in RPMI 1640 medium containing 10 percentfetal calf serum (FCS) together with different concentrations of theappropriate test peptide multimer from the aqueous composition. Thesecells are designated as the effector cells, and are H-2^(d).

Target cells (phytohemagglutinin-stimulated (PHA) blasts of syngeneicmouse spleen cells or P815 mouse cells expressing a corresponding HIVprotein] (1×10⁶/assay sample) are washed with serum-free RPMI 1640medium three times and then admixed, contacted and maintained(incubated) at 37 degrees C. for about 1.5 to about 3 hours togetherwith various concentrations of the test peptide and 300 μCi of sodiumchromate (specific activity 200-500 μCi/mg of Cr, New England Nuclear,Boston, Mass.). The target cells samples are subsequently washed withRPMI 1640 medium containing 10 percent FCS and the appropriate peptide,and resuspended in RPMI 1640 with 10 percent FCS (2×10⁵ cells/ml) anddifferent concentrations of peptide. A 100 μl aliquot of each cellsuspension is added to a well of a 96-well-U-bottom microtiter plate.

A 100 μl aliquot of the appropriate effector cell suspension is added toeach well and a twofold serial dilution made to obtain differenteffector-to-target cell (E:T) ratios. Control wells receive 0.1 ml ofRPMI medium with 10 percent FCS alone in the absence of effector cellsto obtain a value for spontaneous ⁵¹Cr release, and receive 0.1 ml of 5percent Triton X-100 detergent to obtain a value for total ⁵¹Cr release.

The plates are incubated at 37 degrees C. for about 3 to about 4 hours,following which 100 μl of supernatant from each well is monitored in agamma counter to determine ⁵¹Cr release. The percent cytotoxicity iscalculated as$\frac{\left( {{{Effector}\quad {Cell}} - {{Stimulated}\quad {Release}}} \right) - \left( {{Spontaneous}\quad {Release}} \right)}{{{Total}\quad {Release}} - {{Spontaneous}\quad {Release}}} \times 100$

Changes may be made in the construction, operation and arrangement ofthe various parts, elements, steps and procedures described hereinwithout departing from the concept and scope of the invention as definedin the following claims.

What is claimed is:
 1. An isolated peptide of from 7 to about 30 acid residues comprising one or more of the following amino acid residues of gp120 within its structure: AA³⁹⁻⁵¹; AA⁴⁵⁻⁵⁵; AA⁴⁸⁻⁶¹; AA⁷²⁻⁸²; AA⁸¹⁻⁹²; AA⁹²⁻¹⁰²; AA¹⁰⁵⁻¹¹⁶; AA¹¹⁸⁻¹³⁰; AA²⁰⁴⁻²¹⁶; AA²¹⁵⁻²²⁸; AA²⁴⁰⁻²⁵²; AA⁵⁸⁶⁻⁵⁹⁸; AA⁵¹⁹⁻⁵⁴³; or AA⁴¹⁷⁻⁴³⁵.
 2. The peptide according to claim 1 wherein said peptide includes within its structure an amino acid residue sequence, from left to right and in the direction from amino-terminus to carboxy-terminus, represented by a formula selected from the group consisting of: —YLRDQQLLGIWGC—, —FLGFLGAAGSTMGAASLTLTVARQ—, —EQLWVTVYYGVPV—, —VYYGVPVWKEA—, —SVITQACSKVSFE—, and —GVPVWKEATTLFC—.
 3. The peptide according to claim 1 wherein said peptide includes within its structure an amino acid residue sequence, from left to right and in the direction from amino-terminus to carboxy-terminus, represented by a formula selected from the group consisting of: YLRDQQLLGIWGC, FLGFLGAAGSTMGAASLTLTVARQ, EQLWVTVYYGVPV, VYYGVPVWKEA SVITQACSKVSFE, GVPVWKEATTLFC, AHKVWATHACV, CVPTNPVPQEVV, FEPIPIHYCAFPGF, EGCRQIL, ELRSLYNTVAT, VIPMFSALSEG, AMQMLKET, YVDREYKT, KTILKALGPA, and EMMTACQGV.
 4. The peptide of claim 1, wherein said peptide includes within its structure an amino acid sequence of —EQLWVTVYYGVPV—.
 5. The peptide of claim 1, wherein said peptide includes within its structure an amino acid sequence of —VYYGVPVWKEA—.
 6. The peptide of claim 1, wherein said peptide includes within its structure an amino acid sequence of —GVPVWKEATTLFC—.
 7. The peptide of claim 1, wherein said peptide includes within its structure an amino acid sequence of —AHKVWATHACV—.
 8. The peptide of claim 1, wherein said peptide includes within its structure an amino acid sequence of —CVPTNPVPQEVV—.
 9. The peptide of claim 1, wherein said peptide includes within its structure an amino acid sequence of —VLENVTENFNM—.
 10. The peptide of claim 1, wherein said peptide includes within its structure an amino acid sequence of —NNMVEQMHEDII—.
 11. The peptide of claim 1, wherein said peptide includes within its structure an amino acid sequence of —LWDQSLKPCVKLT—.
 12. The peptide of claim 1, wherein said peptide includes within its structure an amino acid sequence of —SVITQACSKVSFE—.
 13. The peptide of claim 1, wherein said peptide includes within its structure an amino acid sequence of —FEPIPIHYCAFPGF—.
 14. The peptide of claim 1, wherein said peptide includes within its structure an amino acid sequence of —GTGPCTNVSTVQC—.
 15. The peptide of claim 1, wherein said peptide includes within its structure an amino acid sequence of —VQCTHGIRPVVSTQ—.
 16. The peptide of claim 1, wherein said peptide includes within its structure an amino acid sequence of —YLRDQQLLGIWGC—.
 17. The peptide of claim 1, wherein said peptide includes within its structure an amino acid sequence of —FLGFLGAAGSTMGAASLTLTVOARO—.
 18. The peptide of claim 1, wherein said peptide includes within its structure an amino acid sequence of —CRIKQIINMWQGVGKAMYA—. 